Reaction mass of 1,3-dioxan-5-ol and 1,3-dioxolan-4-ylmethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The mammalian erythrocyte micronucleus test showed no evidence of a clastogenic response of glycerol formal.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Renowned laboratory following protocol equivalent to OECD guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not specified
- Age at study initiation: 6-8 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: 5 per cage
- Diet (e.g. ad libitum): Porton Combined Diet supplied by BP Nutrition Ltd., Stepfield, Whitam, Essex, Great Britain, ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 29-58
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

None
- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

Not specified

Duration of treatment / exposure:

24 and 48h

Frequency of treatment:

daily

Post exposure period:

None

Remarks:

Doses / Concentrations:
1870 and 3000 mg/kg
Basis:
nominal conc.
For males and females in the First study

Remarks:

Doses / Concentrations:
1620 and 2600 mg/kg
Basis:
nominal conc.
For males in the Second study

Remarks:

Doses / Concentrations:
3125 and 5000 mg/kg
Basis:
nominal conc.
For females in the Second study

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 10 ml/kg

Tissues and cell types examined:

Bone marrows

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In STUDY 1, mice were treated with 80 and 50% of the 7-day maximum tolerated dose (MTD-7) level as a single gavage bolus in corn oil. Due to the high toxicity of these doses towards males, a more accurate determination of the MTD-7 was made separately for males and females in STUDY 2.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A AJOUTER

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were prepared using the paintbrush technique, 24 and 48 hours after a single gavage dose of the test chemical or the positive control agent. Slides were stained with Giemsa and read blind, 1000 polychromatic erythrocytes (PE) being assessed for micronucleated polychromatic erythrocytes (MPE) per animal.

METHOD OF ANALYSIS:
In STUDY 1, cells for assessment were selected from areas of slide rich in PE. As a re-analysis, selected slides from the first study were reassessed for the incidence of MPE. This involved assessment of 10000 PE per slide, reading in continuous fields accross the smear, starting from the beginning of the smear.
In STUDTY 2, 1000 PE were assessed for MPE, starting at the beginning of the smear.
These different methods of slide assessment were due to changes made to Central Toxicology Laboratory test protocol.

Evaluation criteria:

Statistics were the only criteria used to evaluate GF clastogenicity.

Statistics:

Differences between control and treated groups were assessed using single-sided Students t-test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Only mortality was recoded (number of surviving animals/initial number of animals). For males: 4/5 at 1870 mg/kg at 24h sampling; 0/5 at 3000 mg/kg at 24h sampling; 2/5 at 3000 mg/kg at 48h sampling; 4/5 at 1620 mg/kg at 24h and 48h. For females: 4/5 at 1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

FIRST STUDY
Mice were treated with GF at 1870 and 3000 mg/kg. As shown in Table 1 (in "Any other information on results incl. tables"), these dose levels were too much toxic to males.

SECOND STUDY
Due to toxicity met in first study, revised dose levels were employed.

Both studie showed no evidence of clastogenic response of GF. The inactivity of GF was confirmed when selected slides from the first study were assessed more extensively (using 10000 PE rather than 1000 PE) as shown in Table 3.

Statistical evaluation
No statistically significant differences were established between vehicle control and GF treatments groups whereas statistically significant differences were established between vehicle control and cyclophosphamide positive control groups (p < 0.001), using a single-sided Studentst-test (Tables 1 and 2).

Table 1: Incidence of Micronucleated Polychromatic Erythrocytes (MPE) per 1000 Polychromatic Erythrocytes (PE) at the 2 sampling times shown for glycerol formal (GF), corn oil and cyclophosphamide (CP)

 

Treatment	MPE/ 1000 PE ± SD (number of animals)
	24 h sampling		48 h sampling
	Male	Female	Male	Female
Corn oil (10 ml/kg)	3.80 ± 1.1 (5)	2.0 ± 1 (5)	3.80 ± 2.1 (5)	2.20 ± 1.3 (5)
CP (65 mg/kg)	33.20 ± 5.8* (5)	29.80 ± 3.03* (5)	23.0 ± 0.9 (5)	15.20 ± 2.3* (5)
GF (1870 mg/kg)	1.75 ± 0.96 (4)	1.75 ± 0.5 (4)	3.40 ± 0.9 (5)	1.50 ± 1.0 (5)
GF (3000 mg/kg)	-         (0)	2.50 ± 1.3 (4)	3.50 (2)	2.80 ± 1.5 (5)

*Statistically significantly different from controls, p <0.001 (single-sided Student t-test)

 

Table 2: Incidence of MPE/ 1000 PE at the 2 sampling times shown for glycerol formal (GF), corn oil and cyclophosphamide (CP)

 

Treatment	MPE/ 1000 PE ± SD (number of animals)
	24 h sampling		48 h sampling		24 hr/48 h male/female (comnined)
	Male	Female	Male	Female
Corn oil (10 ml/kg)	2.4 ± 1.1 (5)	1.2 ± 1.3 (5)	1.4 ± 1.1 (5)	1.2 ± 0.8 (5)	1.55 ± 1.14 (20)
CP (65 mg/kg)	13.2 ± 1.5* (5)	10.0 ± 1* (5)	15.4 ± 1.9 (5)	10.2 ± 1.8* (5)	13.7 ± 4.2* (20)
GF (1870 mg/kg)	1.25 ± 0.96 (4)		2.4 ± 0.56 (4)		1.34 ± 1.14 (18)
GF (3000 mg/kg)		0.75 ± 0.96 (5)		0.8 ± 1.1 (5)
GF (3000 mg/kg)	2.2 ± 0.84 (5)		1.8 ± 1.6 (5)		1.35 ± 1.34 (20)
GF (3000 mg/kg)	-	1.0 ± 1 (5)		0.2 ± 0.45 (5)

*Statistically significantly different from controls, p <0.001 (single-sided Student t-test)

 

Table 3: Results of re-analysing selected slides from animals included in Table 1, such that 10 000 PE, instead of 1000 PE, were assessed per slide

 

Test group	MPE/ 1000 PE
	Original values (1000 PE)	Original mean ± SD	Values from extended assessment (10 000 PE)	Final mean ± SD
48h, female, vehicle control	4, 1, 2	2.3 ± 1.5	1.6, 1.4, 2.0	1.7 ± 0.3
48h, male, vehicle control	6, 2.6	4.7 ± 2.3	3.3, 2.9, 3.5	3.2 ± 0.3
48h, male, CP	31		18.8
48h, male, GF	4, 4, 4	4 ± 0	3.5, 2.5, 2.7	2.90 ± 0.5

Conclusions:

Interpretation of results (migrated information): negative
The mammalian erythrocyte micronucleus test showed no evidence of a clastogenic response of glycerol formal.

Executive summary:

The mammalian erythrocyte micronucleus test was performed on mice C57BL in 2 times to identify clastogenic potential of glycerol formal (GF).

In the first study, male and female mice were treated with GF at 1870 and 3000 mg/kg by oral gavage route (single dose). Due to high GF toxicity towards males, a second study was performed with the following concentrations in GF: 1620 and 2600 mg/kg for males and 3125 and 5000 mg/kg for females. In both studies, corn oil (at 10 ml/kg) and cyclophosphamide (at 65 mg/kg) were used as vehicle and positive control, respectively respectively

Number of Micronucleated Polychromatic Erythrocytes (MPE) per 1000 Polychromatic Erythrocytes (PE) were assessd at 24- and 48 -hour sampling.

In both studies, no statistically significant differences were established between vehicle control and GF treatments groups whereas statistically significant differences were established between vehicle control and cyclophosphamide positive control groups (p < 0.001), using a single-sided Studentst-test (Tables 1 and 2).

The mammalian erythrocyte micronucleus test showed no evidence of a clastogenic response of glycerol formal.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

According to REACH Annex VIII requirements, the following tests are required:

8.4.1 - an Ames test (OECD 471) is available with a negative result.

8.4.2 - an in vitro cytogenicity study is normally required but does not be conducted if adequate data from an in vivo cytogenicity test are available. Such a test has been performed and follows OECD 474 with a negative result.

8.4.3 - an In vitro V-79 Mammalian Cell Gene Mutation Test (OECD 476) is available with a negative result.

At more, an in vitro test equivalent to OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro) is available with a negative result.

Annex IX requires an appropriate in vivo somatic cell genotoxicity study if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already. As an OECD 474 test is already available (alternative to 8.4.2 in vitro test), there is no other requirement specific to Annex IX

Justification for selection of genetic toxicity endpoint

This Mammalian Erythrocyte Micronucleus Test (OECD Guideline 474) is the most representative one for genetic toxicity.

Justification for classification or non-classification

Glycerol formal shows no evidence of mutagenic activity in the 4 available genetic toxicty tests. Therefore, glycerol formal does not meet criteria for classification